JPH06507565A - IVF methods and devices - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] IVF methods and devices Background of the invention The present invention relates to methods and devices for in vitro fertilization.

Infertility in humans and animals can result from many causes. And that infertility is caused by a lack of sperm. associated with offspring number, low sperm motility, and low percentage of viable motile sperm There are many. One technique to overcome these problems is through in vitro fertilization (rIVF), or That is, the union of sperm and oocytes in a controlled and observable environment outside the body.

As currently practiced, IVF is generally performed using a Percoll gradient or “swim” method. - Unhealthy or non-motile sperm by methods such as Swim-Up J Including pre-separation of healthy motile sperm from (e.g. loafers, sabers, M., M. Publishing, Abreton & 5ink (1990)).

Such a fractionation step may be useful if the sperm are normal (e.g. from the female reproductive system). It is performed in all cases including pathological infertility). Even a normal sperm sample This is due to the fact that it contains a large number of non-motile or dead spermatozoa and epithelial cells. Standards have been established to ensure that these foreign cells cannot interfere with the interaction of motile sperm and eggs. There is. However, in addition to the removal of undesirable spermatozoa, the separation of these spermatozoa The technique also results in a significant loss of motile sperm and therefore It poses its own problems which can lead to failure of IVF.

Once you have obtained the sperm fraction to be used, you can place the sperm into a small number of oocytes (one) in a liquid medium. Generally 1 to 2) is added to the sample dish containing 1 to 2). Animal studies have shown that very small amounts of fluid Injection of semen into the body medium or "microdrops" produces eggs: It is preferred because it maximizes sperm interaction and minimizes the sperm:egg ratio required for fertilization. It's a beautiful thing. (Bavistar, J, Exp, Zoo 1.210:25 9-264 (1979)). However, in human IVF, minute The droplets are exposed to rapid fluctuations in pH and/or changes in molarity due to evaporation. Therefore, such droplets are not used. Therefore, standard IVF methods usually include 2 Fertilization of eggs in milliliters of medium is required. Loafer et al.

These relatively large volumes of medium allow for large numbers of spermatozoa (1 -2XlO' or higher) is required. However, if the sperm sample is abnormal If a large number of oocytes are obtained, or if many oocytes are obtained, losses during sorting may result in There may be an insufficient number of motile sperm for ejaculation. This problem is caused by low sperm count. This is particularly important when the number of motile spermatozoa .

Therefore, in vitro fertilization techniques can achieve high fertilization rates without the need for a pre-fractionation step of spermatozoa. There is a need to provide efficiency. The purpose of the present invention is to meet this need. The purpose is to provide methods and equipment.

Summary of the invention According to the invention, the oocytes to be fertilized are placed around or in a droplet of fertilization medium. High efficiency of fertilization achieved when placed in centrally located individual low volume oocyte chambers be done. The sperm sample, especially the unsorted sperm sample, is then placed in the center of the droplet, or fixed Place it in the hollow surrounding the center. Motile sperm move toward the oocyte in this environment. Therefore, the in5i of motile sperm from non-motile sperm This results in tU separation. Fertilization by sperm entering the oocyte chambers , is extremely easy for low volume chambers.

This fertilization method is preferably carried out in culture dishes specifically designed for this purpose. It will be done. Such culture dishes have multiple oocyte chambers formed on the inner surface of the bottom of the dish. do. Each oocyte chamber exceeds the volume of the oocyte to be fertilized, but It has a small enough volume to be very small. Generally, the oocyte chamber is It has a volume that exceeds the volume by 800% to 2.000%. This oocyte chamber is 0. Around the central circular area of the dish with a diameter of 5 to 3 cm or in the central plateau area May be arranged.

Figure 1 shows the present invention in which an oocyte chamber is formed by modeling the periphery of a microdroplet. An embodiment is shown.

FIG. 2 shows a top view of a culture dish according to the invention.

FIG. 3 shows a cross-sectional view of a culture dish according to the invention.

FIG. 4 shows a sectional view of the T-shaped guide member.

FIG. 5 shows a cross-sectional view of a culture dish according to the invention.

Detailed description of the invention The present invention describes the separation of spermatozoa and isolation of oocytes in a low volume of fertilization medium by a one-step method. Provide both. The efficiency of this method is based on the original fertilization microchamber shown in Figure 1. Explain using.

In FIG. 1, the culture dish is shown from above. This plate has side parts 1 and 1 extending over the golden edge of the plate. It consists of a bottom part 2 and a bottom part 2. A microdroplet of culture medium 3 is placed in the center of the inner surface of the bottom part 2, and the surface of the dish is Form the oocyte chamber 4 by utilizing the adhesive properties of the plastic micropipette. Microdroplets were formed in this shape using a cut tip. Microscopic ring 5 of culture medium A ring of culture medium is placed around each oocyte chamber 4 to form a medium passageway 6. Connected to 5. The dimensions of various parts of the experimental setup are shown in Table 1.

Table 1. Dimensions of the experimental example in Figure 1 To test the efficiency of this design, mouse oocytes (with a diameter of approximately 80 μM) ) was placed in each of the four oocyte chambers formed in a microdroplet of medium, and the undivided Separately frozen-thawed mouse sperm was added to the center of the microdroplet, and an oil droplet was placed over the top surface. held the ground in place. The substance is transferred from the central microscopic area 'a3 to the outside through the passage 6. The flow tends to move the oocyte into the ring 5 of the oocyte chamber. hold in sexual position. Using this design for in vitro fertilization, fertilization efficiency was increased by 5 times. It increased to

The method requires several techniques to manufacture oocyte chambers and passageways and is time consuming. Individual configurations of "custom" IVF devices for each use are therefore impractical.

Furthermore, the imperfectly balanced surface tension between the central microdroplet and the outer ring causes the oocyte to It was found that it can damage cells. Therefore, as embodied in the mouse experiments mentioned above, We designed a culture dish suitable for mass production, in which the principle described above can be implemented more routinely.

FIG. 2 shows a top view of a culture dish specifically adapted for use in the present invention. This culture dish , the two oocyte chambers 4 are placed on opposite sides of the circular area 21 in a microdroplet of medium. It is arranged inside the wall member 22 that defines the edge.

However, the number of oocyte chambers is large and between 2 and 60 are suitable chambers for IVF attempts. It is known that this is within the range of normal oocytes.

The central circular region 21 preferably has a raised portion 32 between the center of the central circular region 21 and the oocyte thin layer. A central depression 31 (FIG. 3) is grown to be formed between the cell chambers 4. central circular area The diameter of the zone 21 is preferably between 0.5 and 30 mm, more preferably between 10 and 20 mm. It is mm.

In use, a microdroplet of culture medium is placed within the wall member 22.

The volume of this microdroplet is determined by the diameter of the central circular region 21, the depth of the central recess 31, and It depends on the volume and number of oocyte chambers 4, but generally there are about 80 or more oocyte cells. and 120 μl.

If a guide member is present to guide the sperm, as described below, the A larger volume may be used. An oocyte 33 is placed in the oocyte chamber 4, and a sperm sample 34 is placed in the oocyte chamber 4. is placed in the center of the central circular area 21. The central circular area 21 is the volume of sperm to be added. may have a ring 24 (Fig. 2) printed in that area to assist in the definition of good. The number of spermatozoa added is generally between 8.000 and 12 in a volume of 8-12 μm. ．． It is 000.

After adding the sperm, the central circular area 21 is closed using a lid 35 that overlaps the wall member 22. may be covered. This dish is then incubated for a predetermined period of time (usually 10 to 20 hours). During this period, healthy motile sperm swim to the oocyte at the edge of the central circular area, where it is deposited. to fertilize the oocytes present in the cells.

The culture dish according to the invention also has a guide member 26 arranged on the surface of the central circular area 21. May have. These guide members are responsible for the presence of the oocyte and therefore for the speed and speed of fertilization. It tends to direct motile sperm toward areas of increased frequency. A preferred guide member is It has a T-shaped cross section (Fig. 4). By using guide members, potentially useful 1-2 m1 is the volume that can be used in "microdroplets". Ru.

The oocyte chamber of the present invention may be a simple depression, as shown in FIG. It may have the shape of a vortex. In the latter case, the oocyte chamber will contain between 200 and 5. OQ Q, preferably 400 to 1.000 microns top diameter, 200 to 2.0 00, preferably from 200 to 500 microns base diameter, and from 150 to i,ooo, preferably having a depth of 150 to 500 microns.

The microchamber shape shown in Figure 1 also allows for e.g. It can also be provided by forming a raised rim on the surface of the dish. oocyte chamber is an annex formed in this raised rim or depression as described above. It's okay.

In addition to the above-mentioned factors that are actively involved in the IVF process, the chamber reduces evaporation. A well may be included around the chamber to provide a source of water vapor.

The grooves are not adjacent to the microchambers, but are located along the microchambers and under the cover.

The method of the present invention performs in vitro fertilization of human oocytes as well as oocytes from animals. It is useful for These oocytes range in diameter from about 80 to about 150 microns. Available in a variety of sizes. For example, human oocytes typically have diameters of about 120 microns, but oocytes from mice and rats are generally about 80 mm in diameter. It's Kron. The term "oocyte" as used herein refers to both oocytes with a small raster of oocytes or surrounding cumulus cells that cannot be separated by It may also include cells.

The oocytes to be fertilized are e.g. The oocyte is manually placed into the oocyte chamber using a micropipette. The oocyte chamber is used for fertilization. It is tailored to the specific species of oocyte being incubated. Avoid damage to oocytes To ensure efficient fertilization, the volume of the chamber is generally 80% of the oocyte volume. 0 to 2. (100%. Chamber size suitable for use with various species, including humans) has dimensions of 200μ diameter x 400μ depth.

The particular medium used in the culture dish is not critical; only one is known to be suitable for a particular species. Any culture medium may be used. For example, using the human medium shown in Table 2 Good results were obtained.

Example 1 Most mouse sperm cannot survive when frozen, so frozen-thawed mouse sperm cannot survive. The effectiveness of the prototype device shown in Figure 1 was studied using a child. This test smell Gorton et al., J. Exp. Zoo 1.239.347 (1986) helps some motile sperm penetrate the oocyte, as described in The oocytes were drilled (zona drilled) and shown in Table 3. Each oocyte was placed in an oocyte chamber formed in a microdroplet of fertilization medium.

A 10 μl sample of frozen-thawed sperm was then placed in the center of the microdroplet. athleticism In five experiments where the proportion of offspring was less than 5%, the mean was 65% (range 53-76%). The fertilization (circle) was achieved.

F-10 Eagle medium (lx) (lx) Component (ig/L) Inorganic salt: CaC1□ (Anhydrous>200.00 CaC12” 2H2044,1-- CuSO4" 5H20' 0.0025 -F e SO4' 7H200, 834--K Cl 2g5.0 400.00 KB2 PO4g3.0 -- Mg Soa’ 7H20152,8200,0ON a Cl 7400. 0 6800.0ON a HCO31200,02200,0ONaHzPO 4”HzO++ 140.0ON a 2 HP 04/7 H20290, OZ　n　S　04　”7　H200,0288Other ingredients: D-glucose 11G0.0 1000. cl.

Hypoxanthine 4.0 - Lipoic acid 0.2 - Phenol red 1.2 to, o.

Sodium pyruvate 110.0 Thymidine 0.7 L-arginine - 17.40 L-7h Gini>EC1211,0- L-asparagine・HzO15,01 L-aspartic acid 13. O L-cystine 25.0 L-cystine -12,00 L-glutamic acid 14.7 L-glutamine 148.0G 292.00 Glycine 7.51 L-Histidine 8.00 L　h　：/HCl　・H2023, OL-isoleucine 2.6 26. 0OL-Leucine 13.0 26.0O L-lysine 29.2O L-lysine HCI 29.0 - L-methionine 7.5O L-phenylalanine 16.5O L-threonine 24.00 L-) Ributofan 4.0O L-tyrosine 18.0O L-tyrosine (disodium salt)- L-valine 23.50 vitamin: Biotin 0.024 1.00 Choline bitartrate l, 00 Choline hydrochloride 0.698 1.00 Folic acid 1.32 1.00 1-Inocital 0.541 2.00 Nicotinamide 1.00 Pyridoxal HCI 1.00 Riboflavin 0.37B 0.10 Thiamin HC11,001,QO D-Ca-pantothenate 0.715 Niacinamide 0.815 Pyrotoxin HCI 0.206 Vitamin B, □ 163B Table 3 NaC1 (v-link Oyto 7851) 5.14g KCI (Baker-$1 -3040) 0.38g KH2PO4 (7 links O-/to $7100) 0.18g Mg5O, -7H20 (Marinckrodt Zero 808B) 0.29g N a HCOi (Fissi Judge S-233) 2.11 g Sodium pyruvate (Schwarzenman Judge 9 04144) 0.04g glucose (Fisher 0-18) 1.00 Grampenicillin G, K salt (Schwartzman Lou 4049) 0.75g streptomycin sulfate (Schwartzmann Mist 3242) 0.05 grams of these ingredients in a flask Add weight and 9951 double distilled H20 and stir until dissolved. Then, Sodium lactate 60%, Funsteel Lab 3.68m17 Eng/L Add 1.00 ml of 1% solution (D7:45358-59). child 3 g/l of crystalline bovine serum albumin (Ventex ll8l-001-3) ) and stir until dissolved.

The medium is filtered through a 0.22 micron Millipore filter for sterilization.

This may be stored at 4°C.

For comparison, band-punctured mouse oocytes were placed in a 2 ml tissue vessel (ti The cells were exposed to the same volume of frozen-thawed spermatozoa in a ssue dish). In this environment , the fertilization rate was probably low because many non-motile spermatozoa blocked access to the oocyte. It was 10% of the total.

The improved efficiency of in vitro fertilization in the culture dish of the present invention is achieved by cryopreservation of spermatozoa and gene transfer. This makes it possible to preserve valuable mouse strains such as strains. Therefore, the original Ming proves a valuable tool in the management of valuable animal strains.

Example 2 Semen from vasectomized male mice that have been proven infertile for more than 6 months The particles were tested in shaped microdroplets as shown in FIG. 30 oocytes in total were placed in the oocyte chamber and a 10 μm sample of unsorted spermatozoa was added. The fertilization medium is The results are shown in Table 3.

In two experiments, including one experiment in which very few free-swimming sperm were present. The fertilization efficiency was 100%.

In contrast, when 106 motile spermatozoa were added to a dish with 2 ml of culture medium, , only 7% of oocytes were fertilized using the same sperm sample.

These results demonstrate that the present invention has a very low percentage of motile spermatozoa, i.e. <10%. It has been shown that fertility can be improved in some cases. The present invention is a method for treating congenital vas deferens. may prove particularly useful in clinical situations such as the repair of a failed or failed vasectomy. . Furthermore, since separation is unnecessary, using the present invention the IVF "procedure" can be simplified.

Example 3 To fertilize oocytes with sperm samples obtained from human patients with congenital absence of vas deferens, The microchamber shown in Figure 1 was used. After collection and washing, this sample is approximately 5% motile. It had 1 x 106 spermatozoa and was heavily contaminated with blood. Two eggs in the micropathology Use the unfractionated washed sample to fertilize the mother cells and barcol the remaining sample. Separated using technology.

After Barcol sorting, motile spermatozoa are used for fertilization of oocytes that are not placed in microchambers. It was not collected. However, one out of two eggs in the microchamber (50%) The embryos were fertilized with unsorted specimens, and fertilized eggs were transplanted.

Example 4 In other studies, standard fertilization was achieved by loading human eggs into the microchamber shown in Figure 1. I did this. Sperm were washed or diluted and added directly to the chamber. Fertility Comparisons were made with normal control fertilization in 2 ml dishes after the "-Up" method. 5ox in ejaculate semen In one patient with to6 motile sperm, the sperm were simply diluted and placed into the microchamber. Arranged. I did a swim-up after washing twice, and this method took about 2 hours. This resulted in a significant loss of sperm. The result is that the microchamber (2/4 of the eggs are fertilized) fertilized) than the control (1/6 fertilized), therefore the microchambers , have shown that the time, expense and loss of sperm associated with washing and sorting can be reduced. There is.

In some additional experiments, sperm were washed and used in the microchamber without fractionation. tiny The fertilization rate of the chamber (9/28.32%) is the same as the fertilization rate of the control vessel (1515B, 27%). The results were similar, indicating that if a microchamber was used again, the need for separation was eliminated.

The foregoing discussion and examples describe the fundamental parameters of the method and apparatus of the present invention. It is. However, it is clear that other shapes can also be used. for example , a central plate surrounded by an annular groove 52 into which sperm are loaded, as shown in FIG. - A culture dish with one or more oocytes 4 arranged on the region 51 is also a subject of the invention. used in law. The central plateau 51 preferably has a lower surface level than the inner surface of the bottom. has.

FIG.1 FIG.3 FIG. 4

Claims (14)

[Claims] 1. (a) Oocytes are placed in an oocyte chamber, and the oocyte chamber is a capacity for the oocytes. the oocyte has a volume 800% to 2000% greater than the located adjacent to, around or in the center of; (b) placing a sperm sample in a portion of the droplet remote from the oocyte chamber; (c) a period sufficient for spermatozoa with common motility to gather in the vicinity of the oocyte chamber; waiting for a period of time and then monitoring fertilization of the oocyte; (d) comprising each step of recovering fertilized oocytes from the oocyte chamber; A method of performing in vitro fertilization with symptoms. 2. Claim 1, characterized in that the droplets have a diameter of 0.5 to 3 cm. The method described in Section 1. 3. Claim characterized in that said spermatozoa are not separated before being placed in said droplet portion. The method according to item 1 or 2. 4. An in vitro fertilization culture dish having a bottom and a side, the bottom of the dish being connected to its inner surface. has multiple oocyte chambers, and each oocyte chamber has a volume of oocytes to be fertilized. The oocyte has a volume of 800% to 2000% greater than 0.5 to 3.0 A culture dish, characterized in that the culture dish is arranged around a central region having a diameter of cm. 5. The oocyte chamber is characterized in that the oocyte chamber is a depression formed on the inner surface of the bottom of the dish. 5. The culture dish according to claim 4, wherein the culture dish has the following characteristics. 6. The central annular region is arranged such that the depth of the liquid disposed in the central annular region is medium than that of the surrounding area. A claim characterized in that it has a depression in its center so that it is deeper in the center. The culture dish according to item 4 or 5. 7. A guide member is formed on the inner surface of the bottom of the dish, the guide member being connected to the central annular It tends to direct the motile sperm placed in the region to swim toward the oocyte. Claims 4 to 6, characterized in that The culture dish according to any one of the items. 8. Claims 4 to 7, characterized in that the oocyte chamber has a vortex shape. The culture dish according to any one of the above. 9. The oocyte chamber has an upper diameter of 200 to 5000 microns, 200 to 2 with a bottom diameter of 1,000 microns and a depth of 150 to 1,000 microns 9. The culture dish according to claim 8, characterized in that: 10. The oocyte chamber has a diameter of about 200 μm and a depth of about 400 μm. The culture dish according to any one of claims 4 to 8, characterized in that: 11. An in vitro fertilization culture dish having a bottom and a side, the bottom having an inner surface thereof. , having an annular groove region defining a central plateau, the plateau extending from the inner surface of the bottom portion; having a low surface level, one or more oocytes are formed on said central plateau, each The volume of the oocyte chamber exceeds the volume of the oocyte to be fertilized by 800% to 2000% A culture dish characterized by having. 12. Particularly, the oocyte chamber is a depression formed on the inner surface of the bottom of the dish. 12. The culture dish according to claim 11, wherein the culture dish has the following characteristics. 13. The oocyte chamber has a diameter of about 200 μm and a depth of about 400 μm. 13. The culture dish according to claim 11 or 12, characterized in that: 14. (a) placing a droplet of fertilization medium in a container; (b) disposing a portion of said droplet at least The said droplet also has a volume smaller than the remaining volume of the droplet outside one oocyte chamber. shaping the droplet to form at least one chamber, the volume of the chamber being 800% to 2000% more than the oocyte volume, (c) spermatozoa with normal motility are placed in said droplet remote from said chamber; When the oocytes are placed in the oocyte chamber, the oocytes are (d) shaping and positioning with respect to the remainder of the droplet; (d) said at least one oocyte chamber; Place the oocyte that can be fertilized inside, (e) a sample of spermatozoa in said droplet located remote from said at least one oocyte chamber; Allocate fees, (f) transporting the spermatozoa into the at least one oocyte chamber, where the spermatozoa have normal motility; remaining in the droplet for a sufficient period of time to collect in the vicinity of (g) fertilizing the oocyte with the sperm and fertilizing the oocyte from the oocyte chamber; Performing in vitro fertilization of oocytes, which is characterized by consisting of each step of recovering oocytes. How to do it.